Methods and reagents for preparing multifunctional probes

Abstract

Multifunctional probes are synthesized in a single step using peptide scaffold-based multifunctional single-attachment-point reagents. To obtain multifunctional probes using the methods of the invention, a substrate (e.g., a nanoparticle, polymer, antibody, protein, low molecular weight compound, drug, etc.) is reacted with a multifunctional single-attachment-point (MSAP) reagent. The MSAP reagents can include three components: (i) a peptide scaffold, (ii) a single chemically reactive group on the peptide scaffold for reaction of the MSAP with a substrate having a complementary reactive group, and (iii) multiple functional groups on the peptide scaffold. The peptide scaffold can include any number of residues; however, for ease of synthesis and reproducibility in clinical trials, it is preferred to limit the residues in the peptide to 20 or less. The reagent can be prepared to yield a predetermined stoichiometric ratio of the functional groups on the scaffold such that the probe has a fixed stoichiometric ratio of the functional groups.

What is claimed is: 
     
       1. A method for preparing a multifunctional probe, the method comprising:
 (a) attaching at least one functional group capable of surviving deprotection to a peptide scaffold in a solid phase; 
 (b) cleaving the peptide scaffold from the solid phase into a solution phase under deprotection conditions; 
 (c) attaching at least one additional functional group to the peptide scaffold in the solution phase under non-deprotection conditions to create a reagent having at least two attached functional groups and a reactive group, wherein the reactive group is either part of the peptide scaffold of step (a) or is attached to the peptide scaffold in a separate step; and 
 (d) attaching the reagent to a substrate by reaction of the reactive group with a complementary reactive group of the substrate. 
 
     
     
       2. The method of  claim 1  wherein:
 the reagent is prepared to yield a predetermined stoichiometric ratio of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) on the scaffold. 
 
     
     
       3. The method of  claim 1  wherein:
 the substrate is selected from nanoparticles, proteins, enzymes, cyclic peptides, linear peptides, antibodies, drugs and vitamins. 
 
     
     
       4. The method of  claim 1  wherein:
 the reactive group of the scaffold and the complementary reactive group of the substrate is a single attachment point for the scaffold and the substrate. 
 
     
     
       5. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a chelate. 
 
     
     
       6. The method of  claim 5  wherein:
 the chelate includes a magnetic material. 
 
     
     
       7. The method of  claim 5  wherein:
 the chelate includes a paramagnetic or superparamagnetic material. 
 
     
     
       8. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) includes a positron-emitting radionuclide or a gamma radiation-emitting radionuclide. 
 
     
     
       9. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is radiopaque. 
 
     
     
       10. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) absorbs photons. 
 
     
     
       11. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a fluorochrome. 
 
     
     
       12. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is selected from proteins, vitamins, enzymes, peptides, antibodies, and drugs that can target a site in a patient. 
 
     
     
       13. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a polymer. 
 
     
     
       14. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a hapten. 
 
     
     
       15. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a charged polymer. 
 
     
     
       16. The method of  claim 1  wherein:
 at least one of the functional group(s) attached in step (a) and the additional functional group(s) attached in step (c) is a dispersion stabilizing agent. 
 
     
     
       17. The method of  claim 1  wherein:
 the reactive group is selected from the group consisting of N-hydroxysuccinimide ester, maleimide, thiol, alkyne, azide, and aldehyde. 
 
     
     
       18. The method of  claim 1  wherein:
 the peptide scaffold includes 20 or less amino acid residues. 
 
     
     
       19. The method of  claim 1  wherein:
 the functional group attached in step (a) is attached to one amino acid residue of the peptide scaffold and the additional functional group attached in step (c) is attached to another amino acid residue of the peptide scaffold. 
 
     
     
       20. The method of  claim 1  wherein:
 the peptide scaffold includes a spacer residue that is not attached to any of the functional groups.